DESCRIPTION: The goal of this research is to increase out understanding of the mechanisms of the ATP-dependent proteolytic enzymes that catalyze the degradation of most proteins in bacterial and eukaryotic cells. These novel enzymes are all large multimeric complexes that cleave proteins in ATP linked reactions. In eukaryotic cells, the major site for protein breakdown is the 20S proteasome, which also generates peptides used in MHC-class I antigen presentation. Unlike conventional proteases, these enzymes degrade proteins completely to peptides before attacking the next substrate molecule. This research will provide understanding of this processive mechanism and determine what factors influence the size and number of peptides generated, and indicate whether the proteases proceed along the substrate in a specific direction. These studies will employ the 20S particles from archaebacteria and mammalian cells, including the modified form of the proteasome (i.e., the "immunoproteasomes") induced by gamma-interferon. The 20S particle serves as the proteolytic core of the 26S proteasome complex, which degrades ubiquitin-conjugated proteins. This complex also contains six different ATPases, and a major goal will be to clarify their functions in protein degradation. Recently a new type of ATP-dependent protease complex (HslU/HslV) from E. coli has been isolated that has homologies to eukaryotic proteasome. The research will elucidate its structure, to clarify how the HslU-ATPase subunits regulate the activity of the HslV peptidase, and to learn if similar complexes are present in other cells or organelles. Finally, the project will explore the processive mechanism of the model ATP-dependent protease, La (lon), which catalyzes the rapid degradation of abnormal proteins in E. coli and mitochondria.